Protein kinases and phosphatases play key roles in regulating biological processes. Coordination of the eucaryotic cell cycle, and transmission of an extracellular stimulus through a cascade of intracellular signal transduction, are two processes which exemplify the importance of protein phosphorylation. The observation that vaccinia virus encodes two protein kinases and a protein phosphatase that are essential for productive viral infection provides compelling evidence that protein phosphorylation plays a major role in regulating the life cycle of this virus as well. At a minimum, it appears that these enzymes play major roles in DNA replication, virion morphogenesis, and early gene transcription. This proposal describes our plans to pursue a thorough molecular genetic analysis of the F10 kinase and H1 phosphatase encoded by vaccinia virus. We have recently mapped a complementation group of temperature-sensitive (ts) mutants to the F10 gene, and determined that non-permissive infections arrest at a very early stage of virion morphogenesis. We have also prepared a viral recombinant in which expression of the H1 gene is dependent upon inclusion of IPTG in the culture medium. In the absence of HI expression, progeny virions are incapable of directing early gene transcription in vivo or in vitro. For both the F10 kinase and H1 phosphatase (aims 1 and 2), we will further characterize the phenotypes engendered by conditional inactivation of the enzyme. We will also identify and characterize their enzymatic substrates and the repertoire of other viral proteins with which they interact. The approaches to be taken include the isolation of additional conditionally lethal viral mutants, selection of second-site suppressors, application of the yeast two-hybrid system and other screens for protein-protein interaction in vivo and in vitro, and testing of numerous viral proteins as substrates. The third aim is focused on a general investigation of viral phosphoproteins. We will attempt to identify virally encoded phosphoproteins within extracts of [32P]-labeled cells and virions. In addition, we will undertake genetic analyses of the roles of F18, 11 and H5, three proteins that appear to be phosphorylated and encapsidated within virions. Finally, we will investigate whether viral infection alters, or is affected by, tyrosine phosphorylation. The genetic and physical autonomy of vaccinia virus from the host cell, the ease with which the viral genome can be manipulated to incorporate altered alleles, and the availability of the complete sequence of the 192 kb vaccinia genome should facilitate our studies.